The proposed research explores molecular approaches for the study and regulation of aberrant metalloenzyme activity in human disease, focusing on the structural and chemical biology of the arginases and the histone deacetylases (HDACs). The arginases utilize a binuclear Mn(II)-Mn(II) cluster for catalysis, whereas the HDACs utilize a single Zn(II) or Fe(II) ion for catalysis. Unexpectedly, these two enzyme families are evolutionarily related and share a common three-dimensional fold despite insignificant amino acid sequence identity and divergent metal ion stoichiometry and selectivity. Increased arginase activity is implicated in cardiovascular disease, asthma, cancer, and parasitic infections, and increased HDAC activity is found in cancer. Both metalloenzymes are validated targets for structure-based drug design. Furthermore, decreased HDAC8 activity is found in Cornelia de Lange Syndrome (CdLS), a congenital birth defect that occurs in one out of 10,000 births. Accordingly, HDAC8 mutants responsible for decreased activity are potential therapeutic targets for molecular activators that can restore normal biological function. To advance our understanding of structure-function relationships in the arginase-deacetylase fold, and to enable innovative molecular approaches for new disease therapies, we will pursue the following lines of investigation: (1) We will fully characterize the reaction kinetics and determine X-ray crystal structures of HDAC8 mutants identified in CdLS. These studies will provide the first molecular view of compromised HDAC8 catalysis underlying the birth defect. Additionally, we will evaluate the ability of molecular activators to restore normal catalytic function in these mutants, and we will determine X-ray crystal structures of HDAC8-activator complexes to delineate their mode of action. (2) We will determine X-ray crystal structures of mutationally inactivated HDAC8 complexed with peptide and/or protein substrates to understand the structural basis of substrate recognition and catalysis. These structures may reveal how some CdLS HDAC8 mutants perturb the enzyme-substrate interface. (3) Finally, we will establish structure-function relationships for parasitic arginases to guide the design of species-specific arginase inhibitors, and we will explore the ability of selected inhibitors to block polyamine biosynthesis in parasites We will also characterize the arginase-like metalloenzyme from Trypanosoma cruzi, which catalyzes an unusual reaction of histidine catabolism.